Opioid drugs such as morphine activate Gi/o-protein-coupled mu-type opioid receptor (mu-OR). mu-opioid agonists produce an array of effects, including analgesia, respiratory depression, sedation, induction of tolerance and dependence, and addiction liability. The magnitude of a drug's maximal effect depends on its intrinsic efficacy, or the degree to which it activates receptors. These studies will investigate the mechanisms of mu-opioid intrinsic efficacy by focusing on cellular signal transduction. In the previous funding period, studies revealed that agonist efficacy to activate G-proteins is dependent on factors including GDP and sodium concentrations, receptor density, receptor-transducer amplification, desensitization and brain region. The major goals of the proposed project are as follows. First, we will test hypothesis that relative efficacy of opioid ligands depends on specific G-alpha subtypes. CHO-K1 cells stably expressing mu-ORs (MOR-CHO) will be co-transfected with pertussis toxin (PTX)-insensitive mutants of different Gi/o subtypes, treated with PTX to eliminate coupling to endogenous Gi-proteins, and the relative efficacies of different full and partial agonists will be determined by [35S]GTP-gammaS binding and adenylyl cyclase inhibition. Ligands will be ranked according to maximal stimulation relative to a standard full agonist to distinguish changes in the magnitude or rank order of ligand efficacy. Differences among mutant G-alpha subtypes will be confirmed by expression of the wild-type G-alpha subunits and by knockdown with RNA interference. Results of these studies will be used to guide studies of opioid efficacy in the brain using receptor and G-protein autoradiographic approaches. The second aim is to test the hypothesis that relative efficacies of mu opioid ligands differ between G-alpha and G-beta gamma regulated effectors. These studies will compare the relative efficacies of these ligands for inhibition of adenylyl cyclase by G-alpha versus activation of mitogenic kinases by G-beta gamma in intact MOR-CHO cells. The relative efficacies of these ligands for sustaining activation of mitogenic kinases over 24 hr will then be compared to their relative efficacies for stimulating cell proliferation, and their effects on programmed cell death and cell cycle progression will be determined. Differences in ligand efficacy for acute versus sustained effector activation due to differences in mu-OR desensitization will be examined, and differences in mechanisms of mitogenesis versus cytotoxicity by different ligands will be investigated with pharmacological inhibitors and dominant negative mutants of specific signaling proteins. By elucidating the mechanisms of opioid ligand efficacy and ligandselective/ mu-OR signaling, these studies may provide new directions in opioid pharmacotherapeutics.